Structure of the Bacillus cell fate determinant SpoIIAA in phosphorylated and unphosphorylated forms

BACKGROUND: The asymmetric cell division during sporulation in Bacillus subtilis gives rise to two compartments: the mother cell and the forespore. Each follow different programs of gene expression coordinated by a succession of alternate RNA polymerase sigma factors. The activity of the first of these sigma factors, sigmaF, is restricted to the forespore although sigmaF is present in the predivisional cell and partitions into both compartments following the asymmetric septation. For sigmaF to become active, it must escape from a complex with its cognate anti-sigma factor, SpoIIAB. This relief from SpoIIAB inhibition requires the dephosphorylation of the anti-sigma factor antagonist, SpoIIAA. The phosphorylation state of SpoIIAA is thus a key determinant of sigmaF activity and cell fate. RESULTS: We have solved the crystal structures of SpoIIAA from Bacillus sphaericus in its phosphorylated and unphosphorylated forms. The overall structure consists of a central beta-pleated sheet, one face of which is buried by a pair of alpha helices, while the other is largely exposed to solvent. The site of phosphorylation, Ser57, is located at the N terminus of helix alpha2. The phosphoserine is exceptionally well defined in the 1.2 A electron density maps, revealing that the structural changes accompanying phosphorylation are slight. CONCLUSIONS: Comparison of unphosphorylated and phosphorylated SpoIIAA shows that covalent modification has no significant effect on the global structure of the protein. The phosphoryl group has a passive role as a negatively charged flag rather than the active role it plays as a nucleus of structural reorganization in many eukaryotic signaling systems.     

Mutational analysis of RsbT, an activator of the Bacillus subtilis stress response transcription factor, sigmaB

SigmaB, the stress-activated sigma factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for sigmaB induction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteen rsbT mutations blocked sigmaB induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was sigmaB induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate sigmaB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.     

Efficient regulation of sigmaF, the first sporulation-specific sigma factor in B.subtilis

Differential gene expression is established in the prespore and mother-cell compartments of Bacillus subtilis through the successive activation of a series of cell-type-specific sigma factors. Crucial to the success of this process is the control of the first prespore-specific sigma factor, sigmaF. sigmaF is regulated by the proteins SpoIIAB, SpoIIAA and SpoIIE. SpoIIAB forms an inhibitory complex with sigmaF, which can be dissociated by interaction with SpoIIAA. During this interaction SpoIIAA is phosphorylated. SpoIIE is a membrane-bound phosphatase that dephosphorylates SpoIIAA, thereby re-activating it. It is not understood how sigmaF is activated specifically in the prespore but not in the mother cell. Here, we use a recently developed fluorescence spectroscopy technique to follow in real time the formation of sigmaF.SpoIIAB complexes and their dissociation by SpoIIAA. We show that complete activation of sigmaF is induced by a tenfold increase in SpoIIE activity. This result demonstrates that relatively small changes in SpoIIE activity, which could arise from asymmetric septation, can achieve the all-or-nothing response in sigmaF activity required by the cell. For long-term sigmaF activation, we find that sustained SpoIIE activity is required to counteract the activity of SpoIIAB. Even though the continual phosphorylation and dephosphorylation of SpoIIAA by these two enzymes will expend some ATP, the formation of SpoIIAA.SpoIIAB.ADP complexes greatly diminishes the rate of the phosphorylation reaction, and thus minimizes the wastage of energy. These features provide a very efficient system for regulating sigmaF.     

Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis

In the pathway that controls sigmaB activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of sigmaB of Bacillus subtilis. We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS. RsbT is then free to interact with and activate RsbU, which in turn ultimately activates sigmaB. In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues. The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS. However, it appeared that RsbT preferentially phosphorylates T171. We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205.     

A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins

Regulation of expression of the general stress regulon of Bacillus subtilis is mediated by the activation of the alternative sigma factor sigmaB. Activation of sigmaB is accomplished by a complex regulatory network involving protein-protein interactions and reversible protein phosphorylation. PSI-BLAST searches were performed and phylogenetic trees for sigmaB and its regulatory proteins were constructed. Occurrence of sigmaB is restricted to a small group of gram-positive bacteria (Bacillus, Staphylococcus, Listeria). Related sigma factors also involved in stress responses are present in Mycobacterium tuberculosis, Streptomyces species and even in cyanobacteria (Synechocystis species). Putative regulatory proteins found in several other bacterial species can be broadly catagorized into three categories: Anti sigma factors, anti-anti sigma factors and phosphatases. Anti sigma factors are able to bind to sigma factors and are also kinases of anti sigma factor antagonists. Only in their nonphosphorylated state, these antagonists are able to bind to the anti sigma factor. Phosphorylated antagonists can be dephosphorylated by PP2C phosphatases. These phosphatases are of pivotal importance for activation of the sigma factor. Different phosphatases identified in this search contain a wide variety of domains found in signal transducing proteins (PAS/PAC, GAF, REC, HATase_c, HAMP). The HATPase_c domain found in several phosphatases most probably constitutes a serine/threonine kinase domain of anti sigma factors. Such proteins are most probably bifunctional anti-anti sigma factor kinases and phosphatases. The regulatory network of anti-anti sigma factors anti sigma factors and phosphatases is probably ancient and most likely evolved from a structurally similar network found in the Deinococcus radiodurans genome. In completely sequenced genomes of several bacterial species, some elements of the network are missing. The N-terminus of RsbU, a phosphatase activated in response to environmental stress exhibits similarities to a region in the beta chain of phenylalanyl-tRNA synthetases.     

Identification of sigmaB-dependent promoters using consensus-directed search of Streptomyces coelicolor genome

SigmaB plays an important role in both osmoprotection and proper differentiation in Streptomyces coelicolor A3(2). We searched for candidate members of the sigmaB regulon from the genome database, using the consensus promoter sequence (GNNTN14-16GGGTAC/T). The list consists of 115 genes, and includes all the known sigmaB target genes and many other genes whose functions are related to stress protection and differentiation.     

Proteome signatures for stress and starvation in Bacillus subtilis as revealed by a 2-D gel image color coding approach

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation.